Bottom Line:
The vast majority of annotated transcripts in bacteria are mRNAs.Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli.We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene.

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA.

ABSTRACTThe vast majority of annotated transcripts in bacteria are mRNAs. Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli. We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene.

f2: (A) Expression of a lacZ reporter gene fused to putative aRNA promoters. Wild-type (gray, right) or mutant (orange, right; −10 hexamers replaced by GGGCCC) aRNA promoter regions (200 bp upstream to 10 bp downstream of +1) were transcriptionally fused to lacZ on a single-copy plasmid (a derivative of pBAC-BA-lacZ, Addgene plasmid 13423, in which the HindIII-NotI fragment was replaced with an E. coli rRNA transcription terminator). β-Galactosidase assays were performed using E. coli MG1655 ΔlacZ. Gene names indicate the overlapping protein-coding genes. Numbers in parentheses indicate the number of times the aRNA 5′ end was sequenced/the number of base matches to the −10 hexamer consensus. Note that one promoter tested (eutB) is located in an untranslated region between the eutB and eutC genes (transcribed within an operon), but the putative RNA overlaps the eutB gene. There is no correlation between the number of sequence reads and promoter strength. We speculate that this is due to a combination of differential aRNA stability, introduction of bias by the PCR step of library construction, and the known sequence bias of RNA ligase T4 Rnl1 (27). wt, wild type. (B) Expression of a lacZ reporter translationally fused to rplJ or yrdA, including the natural rplJ or yrdA protein-coding gene promoter, on a single-copy plasmid (described above). Expression levels were measured for wild-type (gray, right) and mutant (orange, right) aRNA −10 hexamers/+1 transcription start sites (mutations did not alter the protein-coding sequence of the mRNA and did not substantially alter the codon bias; the rplJ aRNA −10 hexamer mutated from TACAGT to GACGGT, and the +1 transcription start site mutated from A to G; the yrdA aRNA −10 hexamer mutated from CATAAT to CGTAGT, while the +1 transcription start site was unchanged [boldface shows change]). Expression of rplJ::lacZ and yrdA::lacZ was measured using MG1655 ΔlacZ and MG1655 ΔlacZ ΔyrdA, respectively.

Mentions:
To experimentally validate the putative aRNAs, we fused the promoter regions (up to 200 bp upstream of the putative transcription start site) of 10 aRNAs to a lacZ reporter gene and measured expression levels in a β-galactosidase assay. In 9 out of 10 cases tested, we detected lacZ expression that was significantly reduced by mutation of the −10 hexamer (Fig. 2A). We conclude that the large majority of putative aRNAs are genuine and that our transcription start site assignments are highly accurate.

f2: (A) Expression of a lacZ reporter gene fused to putative aRNA promoters. Wild-type (gray, right) or mutant (orange, right; −10 hexamers replaced by GGGCCC) aRNA promoter regions (200 bp upstream to 10 bp downstream of +1) were transcriptionally fused to lacZ on a single-copy plasmid (a derivative of pBAC-BA-lacZ, Addgene plasmid 13423, in which the HindIII-NotI fragment was replaced with an E. coli rRNA transcription terminator). β-Galactosidase assays were performed using E. coli MG1655 ΔlacZ. Gene names indicate the overlapping protein-coding genes. Numbers in parentheses indicate the number of times the aRNA 5′ end was sequenced/the number of base matches to the −10 hexamer consensus. Note that one promoter tested (eutB) is located in an untranslated region between the eutB and eutC genes (transcribed within an operon), but the putative RNA overlaps the eutB gene. There is no correlation between the number of sequence reads and promoter strength. We speculate that this is due to a combination of differential aRNA stability, introduction of bias by the PCR step of library construction, and the known sequence bias of RNA ligase T4 Rnl1 (27). wt, wild type. (B) Expression of a lacZ reporter translationally fused to rplJ or yrdA, including the natural rplJ or yrdA protein-coding gene promoter, on a single-copy plasmid (described above). Expression levels were measured for wild-type (gray, right) and mutant (orange, right) aRNA −10 hexamers/+1 transcription start sites (mutations did not alter the protein-coding sequence of the mRNA and did not substantially alter the codon bias; the rplJ aRNA −10 hexamer mutated from TACAGT to GACGGT, and the +1 transcription start site mutated from A to G; the yrdA aRNA −10 hexamer mutated from CATAAT to CGTAGT, while the +1 transcription start site was unchanged [boldface shows change]). Expression of rplJ::lacZ and yrdA::lacZ was measured using MG1655 ΔlacZ and MG1655 ΔlacZ ΔyrdA, respectively.

Mentions:
To experimentally validate the putative aRNAs, we fused the promoter regions (up to 200 bp upstream of the putative transcription start site) of 10 aRNAs to a lacZ reporter gene and measured expression levels in a β-galactosidase assay. In 9 out of 10 cases tested, we detected lacZ expression that was significantly reduced by mutation of the −10 hexamer (Fig. 2A). We conclude that the large majority of putative aRNAs are genuine and that our transcription start site assignments are highly accurate.

Bottom Line:
The vast majority of annotated transcripts in bacteria are mRNAs.Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli.We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene.

Affiliation:
Wadsworth Center, New York State Department of Health, Albany, New York, USA.

ABSTRACTThe vast majority of annotated transcripts in bacteria are mRNAs. Here we identify ~1,000 antisense transcripts in the model bacterium Escherichia coli. We propose that these transcripts are generated by promiscuous transcription initiation within genes and that many of them regulate expression of the overlapping gene.